The rapid spread of multidrug-resistant strains of common bacteria pathogens pose a significant threat to public health, calling for an accelerated effort to develop novel antibiotics acting on previously unexplored targets. Most notably, many strains of Staphylococcus aureus, a major source of infections in hospitals, are resistant to commonly used antibiotics. Comparative genomics analysis and gene essentiality studies implicated nicotinic acid mononucleotide (NaMN) adenylyltransferase of the NadD family, the key indispensable enzyme in NAD biogenesis of most bacteria (including S. aureus), as a prominent target for the development of novel antimicrobial agents. In previous studies, first, small molecule inhibitors of NadD enzymes from representative gram-positive and gram-negative bacteria were identified, and then their on- target antibacterial activity was confirmed. Some of the identified compounds also showed strong inhibitory activity against S. aureus NadD enzyme. These results provided validation of the NadD target and set the stage for searching for new inhibitors with substantially improved affinity, selectivity, and antibacterial properties. This goal would be addressed in the proposed project by pursuing the following specific aims: (I) screen the MLPCN small molecule compound library and perform hit optimization to identify high-affinity inhibitors of S. aureus NadD enzyme (with IC50 d1 microM); (II) test confirmed hits by secondary assays and in the counter screen against is functional but structurally distinct human enzyme; (III) assess antibacterial properties and kinetic mechanisms of selected nominated probes. The feasibility of the proposed project is supported by the availability of the pure recombinant target enzyme as well as HTS assay methods developed in preliminary studies. Selected best inhibitors will be used in future work as molecular probes to further explore NAD biosynthesis as a target pathway for the development of novel antibiotics.